Process for production of streptomycylamines



each .1 7;

Patented Dec. 29, 1953 PROCESS FOR PRODUCTION OF .S'IREPTOMYGYLAMINESWalter A. Winston, Forest Hills, N. Y-., assignor to Food ResearchLaboratories, Inc, Long ls- "land Gity, N. Y., a corporation of New YorkNo Drawing. Application May 26, 1950, Serial No. 164,597

3 Claims. (01. 260-210) This invention relates to novel derivatives ofstreptomycin, and .in particular .is directed to the discovery of uniquepropertiesof N-substituted streptomycylamines.

In my pending application for Letters Patent,

Serial No. 43,349, I have described .novel derivatives of streptomycincharacterized by the structure:

Strep. CHQNHR:

wherein the symbol Strep designates the portion of the streptomycinmolecule linked to its aldehyde group --'CHO, and R designates asubstituent such as aliphatic having at least 3 carbon atoms, aromaticor a combination thereof. The compounds therein describedwe're found topossess RF values that are markedly higher than the RF value of thearent molecule streptom cin.

In continuing the investigations of this unique class of compounds Ihave "found that a particular segment thereof possesses unusualbacterieidai and fungicidal roperties, such properties beingsurprisingly limited to the compounds of the type structure abovedescribed wherein R designates the radicals in the range from aboutseven carbon atoms 'to sixteen carbon atoms.

- The actual range for a specific organism may be somewhat narrower thanthe indicated range.

In the following examples and tables there are set forth the results ofantibacterial and anti-.

fungal experiments carried out with a variety of organisms.

EXAMPLE -1 antibiotic activity of sft'reptomycyicmines against .Amethanol solution (200 mil) of the calcium chloride double salt ofstreptomycin trihydrochloride (14.9 gni-. 0.01 mole) and n-decylamine(9.4 g., 0.06 mole) was reduced under 1000 p. s. i. of hydrogen at 80435in the presence of 5 per cent palladium catalyst (5 -gm.:).' After sixhours, heating was discontinued and the hydrogenation was allowed toproceed 18 hours. The

catalyst was filtered off, washed with methanol 1 and the combinedfiltrate and washings were poured into 10 volumes of absolute other withstirring. The precipitate was filtered off, washed with ether anddissolved in distilled water. The solution was adjusted from pH 8 to pH5.5 with hydrochloric acid, frozen and lyophilized, and there wasobtained the N-n-decyl and strepto- ,mycylam-ine a yield of 13.2 grams.

The crude product was purified via the tetrahelianthate route from whichthe pure tetrahydrochloride was obtained by the method of purisomeprecipitation occurred which made reading fying streptomycin describedby Folkers et al, J. American Chem. Soc, 68, 1460 (1946) in similarmanner there were repared the other members of this class of compoundsrunhing from the N-n-propyl streptomycylamine to the N -n-octadecylstreptomyoylamine.

The antibiotic activity of these compounds against E. coli wasdetermined by the United States Food and Drug Administration test il ed-'e'ral Register, 12, 22-244, (April-4,1947) 2 NOTE.As an example, thisdefinition of activity may be better understood by noting that 1,000 'yof streptomycin-3H0! has anectivity of 841 7 of streptomycin whereas1,000 y or the N -n-propyl streptomycylamine has only the equivalentactivity of 'v of strepto mycin.

3 Impure compounds.

EXAMPLE 2 Action of ill-substituted streptomycy'lominee against fungi(a) Monilia albicans ATCC 2091 In test tubes containing '8 cc. ofsterile mycophil broth there were added 1 m1. of sterile human bloodserum (or water as indicated below), and 1 ml. (or less as indicatedbelow) of a 0.2% solution (sterilized by boiling) of the substance to betested. There was also added 0.1 ml. of a 48 hour culture of M. alb'z'cots (American Type Culture Collection (ATGC) 209.1) grown at 30 C. Theincubation of the tubes was carried out'u to four days at 30 C. and thegrowth was observed. At the end of two days, however, the materials weresubcultured into plain mycophil broth and incubated and observed afterone and three days at 30 C.

On adding the compounds to the original media of the growth a littledifficult; and hence, recourse to sub-culture was deemed expedient. itis to be noted that inevaluat'ing the streptomycylamines, agar whichinhibits the action 3 thereof should not be present. The agar plate testtherefore cannot be used.

In this example the following compounds were tested. and are designatedby the compound numbers indicated.

Compound No.

Nn-hutyl streptomycylamine N-n-hexyl streptomycylamine N-n-heptylstreptomycylamine N-n -octyl streptomycylamine N-n-dccylstreotomycylamine N-n-dodecyl strentomycylamine N-n-tetradecylstreptomycylamine N-n-hexadecyl streptomycylamine. N-n-octadecylstreptomycylamine TABLE II [48 hour subculture read 24 hours aftersubculture] Compound No.

Ml. of 0.2% solution in original tube tubes containing the antibioticswere read with.

some difficulty, due to the precipitate formed on adding the compoundsto the medium. The results thereof are shown in the following table.

TABLE III, [Tubes read after 4 days in the presence of the antibiotic}Compound N0. M1. of 0.2% solution in original tubes 1 In this and thefollowing tables indicates difficulty in deciding. by gross inspection,whether the organism was present or not.

It is evident that even the lowest concentrations of compounds '7, 8 and9 possess inhibitory powers. Of these, 7 and 8 are better than 9 sinceon sub-culture (see Table II) 9 failed to reduce the count as much as 7and 8. For this test the peak activity is probably given byN-ntetradecyl streptomycylamine and the corresponding N-n-hexadecylderivative.

The fact that these compounds, i. e., the most eifective members of thegroup are active against M. albicans even in th presence of human serumwhich inhibits most fungistatic and fungicidal agents, is of greatsignificance and importance. These compounds are accordingly of greatvalue for topical use against monilial vulvo vaginitis and otherinfections due to monilia.

The fungicidal activity of these streptomycylamines against Moniliaalbicans is further demonstrated in the following table. To five ml. ofa solution of N-n-dodecyl streptomycylamine in water, 0.5 ml. of a 48hour culture of M. albicans was added. After minutes and minutes at 37C. subcultures were made and read after two days at C.

TABLE Iv Concentration of N-n-dodecyl strep- Time tomycylamine Sminutes15 minutes When streptomycylamine and dihydrostreptomycin were used astest substances in the same way, those antibiotics failed to inhibit atall.

In a similar test the following compounds gave results set forth in thetable below:

TABLE v Time Compounds and concentrations 5min. 15min.

N-n-decyl streptomycylamine:

+ E l i It will be observed that the N-n-dodecyl and the N-n-hexadecylderivatives are the best of the straight chain types. The N-n-propylappears to be ineffective in the test. It also will be noted that themolecular size of the substituent is important. Thus when thesubstituent contains an alicyclic group it should be linked to arelatively long chain. Thus the cyclohexyl derivative inhibits where thecyclohexyl ethyl derivative does not inhibit.

(b) Tricophyton Interdigital ATCC 9375 A 10 day old culture of theorganism in mycophil broth was shaken with beads and filtered through acoarse filter aseptically. 0.1 ml. was used as the inoculum for tubescontaining 8 ml. of mycophil broth, 1 ml. of sterile human plasma (or 1ml. of water), and 1 ml. or less of an 0.2% solution of the compoundsnumbers 1-9, as designated above. While the addition of the solutions ofthe compounds=caused precipitates to form in the medium this did notinterfere as much as it did in the tests with M. albicans because themycelial growth of the Tricophyton is easily recognized.

The results of this test are given in the following table, the readingshaving been made at th end of 7 days, and at 30 C.

TABLE VI Compound Nos. M]. of 0.2% solution of compounds 1 g 1 2 a 4 a a7 i s 5 9 l i 9 .1 t

The tubes were also read after 10 days. Negative tubes were subculturedand the subcultures were read after an additional 10 days. The resultsof this phase of the test are shown in the following table.

TAB LE VII 10 day readings in tubes Compound Nos.

Subculture of 0.5

Ml. of 2% solution:

01 Subculture of 0.1

1 A bacterial contamination was observed.

It will be noted that 0.5 ml. of the 0.2% solution (in about 10 ml. oftest solution) of com pounds 3 through 8 inhibit Tricophytoninterdigital. It will also be noted that a subculture of the organismfails to grow out when taken from previously negative tubes showing thatin the presence of serum in this test the compounds are fungicidal aswell as fungistatic. This is in contrast to the action on M. Albioanswhere in that test only a fungistatic action is observed. The peakactivity against Tricophyton appears to be within the range of theN-n-heptyl streptomycylamine through the N-n-hexadecylstreptomycylamine. A further test was carried out in the investigationof the properties of these streptomycylamines. The technique employedwas that of standard phenol coeiiicient test of the U. S. Food and DrugAdministration Bulletin 198 but modified as to medium and organism used.

A 4% solution of each compound was sterilized by boiling. Then 2.5 cc.of each solution was diluted with 2.5 cc. of water or 2.5 cc. of 20%sterile human blood serum. In another set of experiments there was addedto 0.25 ml. of each test solution 4.75 cc. of Water or 2.25 cc. of waterplus 2.5 cc. of 20% human blood serum. To each tube there was added 0.5cc. of filtered culture (10 day cultures of mycophil broth shaken withbeads and filtered through coarse sterile filters).

In the tests all the tubes were held at 37 C. in a water bath. After 5minutes and 15 minutes of contact time, transplants to plain sterilemycophil broth were made. The transplants were incubated for days at 30C. and read. The results of these experiments are set forth in thefollowing table.

TABLE VIII Compound and concentration 5 min. 15 min.

Ill]

2% (serum) 0.2% (serum) N-n-decyl streptomycylam [III 2% (serum) .l

0.2% (Scrum) N-n-hexadecyl streptomycylamine:

l lll 1 A bacterial contamination was observed.

2 This anomalous result, at high concentration as contrasted with the0.2% below which cause inertia, appears to be due to the presence of acontaminant.

These results show that even in the presence of serum the compoundstested will kill in 5 to 15 minutes at one or both of the concentrationsemployed. It will be noted that the N-S- cyclohexyl hexylstreptomycylamine and the N-n-decyl kill sooner and at lowerconcentrations.

It will be understood that the foregoing description of this inventionis merely illustrative of its principles, and, accordingly, the appendedclaims are to be construed as defining the invention Within the fullspirit and scope thereof.

I claim:

1. Method of preparing compounds of the formula StrepCH2-NHR whereinstrep designates the entire molecular structure of streptomycin exceptthe aldehydic radical thereof, and R designates an aliphatic hydrocarbonradical of from 7 to 16 carbon atoms, which comprises mixing amethanolic solution of streptomycin trihydrochloride calcium chloridedouble salt with a primary aliphatic amine, having from 7 to 16 carbonatoms, and subjecting said mixture to hydrogenation at a temperature ofabout -85 C. with hydrogen under a pressure of about 1000 p. s. i. inthe presence of a hydrogenation catalyst.

2. Method in accordance with claim 1 wherein the temperature of 80-85 C.is maintained for about 6 hours.

3. Method in accordance with claim 2 wherein the hydrogenation, aftercessation of the heating to 80-85 C., proceeds for about 18 hours.

WALTER A. WINSTEN.

N 0 references cited.

1. METHOD OF PREPARING COMPOUNDS OF THE FORMULA STREP-CH2-NHR WHEREIN"STREP" DESIGNATES THE ENTIRE MOLECULAR STRUCTUE OF STREPTOMYCIN EXCEPTTHE ALDEHYDIC RADICAL THEREOF, AND R DESIGNATES AN ALIPHATIC HYDROCARBONRADICAL OF FROM 7 TO 16 CARBON ATOMS, WHICH COMPRISES MIXING AMETHANOLIC SOLUTION OF STREPTOMYCIN TRIHYDROCHLORIDE CALCIUM CHLORIDEDOUBLE SALT WITH A PRIMARY ALIPHATIC AMINE, HAVING FROM 7 TO 16 CARBONATOMS, AND SUBJECTING SAID MIXTURE TO HYDROGENATION AT A TEMPERATURE OFABOUT 80*-85* C. WITH HYDROGEN UNDER A PRESSURE OF ABOUT 1000 P. S. I.IN THE PRESENCE OF A HYDROGENATION CATALYST.